Human erythrocyte membranes have at least three electrophoretically distinguishable glycoprotein compounds. Glycoproteins are proteins conjugated with a small number of heterosaccharide prosthetic groups. Electrophoretic and immunologic analysis of erythocyte membrane glycoproteins reveal that different glycoproteins are associated with different blood group antigens (ABO, MN, S and others).
The major glycoprotein of the human red cell membrane, called glycophorin A, is a single polypeptide chain composed of one hundred and thirty-one amino acid residues and approximately one hundred and twenty-five sugar residues. Glycophorin molecules are distributed uniformly over the red cell surface and appear to be associated with the intra-membranous particles. Glycophorin A occurs in two allelic forms responsible for the MN blood group, as well as serving as a receptor for kidney bean phytohemagglutinin, wheat germ agglutinin and influenza viruses. (An allele is either of a pair of genes located at the same position on both members of a pair of chromosomes which convey inherited characteristics alternatively in accordance with Mendelian laws.)
MN is a blood group system in which two alleles identified as M and N determine the presence of corresponding antigens on the red cells and give rise to three phenotypes MM, MN and NN. Humans may be "typed" as having either the M-type (MM homozygous), or the N-type (NN homozygous) or an MN combination type (MN heterozygous) of the two genotypes on their red cells.
The two alleles, M and N, determine a difference in the amino acid sequence of glycophorin A, such that glycophorin A normally occurs in two forms, glycophorin A.sup.M and glycophorin A.sup.N. Glycophorin A.sup.M differs from glycophorin A.sup.N in only two amino acid residues. Serine and glycine are found in positions 1 and 5 of the amino acid sequence of glycophorin A.sup.M and leucine and glutamic acid replace these amino acids at the corresponding positions in glycophorin A.sup.N. It has been postulated that the serological distinction between A.sup.M and A.sup.N resides within the amino terminal regions of these proteins.
Blood group typing has, up until now, been done by obtaining from rabbit antisera, antibodies (immunoglobulins) and reacting them with the blood sample to be typed. Conventionally, this is done by repeated injections of human red blood cells into the animal at two- or three-week intervals, bleeding the animal, isolating the serum and absorbing out the cross-reacting antibodies. Based on their ability to agglutinate red blood cells, clinical laboratories use this type of antisera to type blood samples. Antisera produced in this manner, typically contain polyclonal mixtures of antibodies produced by the animal in response to different antigens or to different immunologic determinants on the same antigen and include antibodies that differentiate between the two glycophorins on the basis of differences in the amino acid residues, either at the first or at the fifth position or at both positions. Antisera are not precisely defined chemical reagents and their composition, therefore, vary from lot to lot of the serum.
For some applications, this lack of specificity or precision is not crucial. For certain other applications, specific monoclonal antibodies would be a critical requirement. Monoclonal antibodies to some specific antigens have been prepared by fusing antigen-sensitized mouse spleen cells (capable of producing antibodies to the specific antigen) with mouse myeloma cells (with self-replicating characteristics both in vivo and in vitro) and cloning the hybrid myeloma cells which now produce the desired antibodies in large quantities.
However, deriving a specific hybrid myeloma that produces antibodies to a selected antige or to a specific antigenic determinant is usually fraught with difficulties as the characteristics of the hybridoma vary with the antigenic determinant unique to the antigen and with the immune response of the animal chosen. Furthermore, when the immune response is very weak, the search for a hybrid clone secreting the specific antibody among the many clones secreting nonspecific immunoglobulins present special problems and require special procedures.
Preparation of monoclonal antibodies for sheep red blood cells, by fusing a mouse spleen cell sensitized with sheep red blood cells, with a mouse myeloma cell and culturing the fused hybrid cell (hybridoma) (which now produces antibodies to sheep red blood cell antigens), in a suitable culture medium, was first reported by Milstein and Kohler in Nature, 256, 493 (1975) and in Eur. J. Immunol. 5, 720 (1975). Koprowski et al., (U.S. Pat. No. 4,196,263) disclosed a method and cell line for the production of anti-influenza antiviral antibodies and Wands et al., (U.S. Pat. No. 4,271,145) described a cell line for the production of monoclonal antibodies to hepatitis virus.
But few monoclonal antibodies have been developed or reported for blood group substance antigens or for the surface proteins of red blood cells. It would be highly desirable, therefore, to obtain monoclonal antibodies to specific blood group substances for utilization as reliable standardized reagents for accurate clinical blood-typing for transfusions and for other clinical and nonclinical uses. Monoclonal antibodies specific for the M and N form of human glycophorin A would be superior blood-typing reagents since they can be obtained in large quantities in pure form eliminating lot to lot varibility and the need to absorb whole sera in order to obtain specific reagents.